Molecular mechanisms underlying NLRP3 inflammasome activation and IL-1β production in air pollution fine particulate matter (PM2.5)-primed macrophages

Exposure to air pollution fine particulate matter (PM2.5) aggravates respiratory and cardiovascular diseases. It has been proposed that PM2.5 uptake by alveolar macrophages promotes local inflammation that ignites a systemic response, but precise underlying mechanisms remain unclear. Here, we demonstrate that PM2.5 phagocytosis leads to NLRP3 inflammasome activation and subsequent release of the pro-inflammatory master cytokine IL-1β. Inflammasome priming and assembly was time- and dose-dependent in inflammasome-reporter THP-1-ASC-GFP cells, and consistent across PM2.5 samples of variable chemical composition. While inflammasome activation was promoted by different PM2.5 surrogates, significant IL-1β release could only be observed after stimulation with transition-metal rich Residual Oil Fly Ash (ROFA) particles. This effect was confirmed in primary human monocyte-derived macrophages and murine bone marrow-derived macrophages (BMDMs), and by confocal imaging of inflammasome-reporter ASC-Citrine BMDMs. IL-1β release by ROFA was dependent on the NLRP3 inflammasome, as indicated by lack of IL-1β production in ROFA-exposed NLRP3-deficient (Nlrp3−/−) BMDMs, and by specific NLRP3 inhibition with the pharmacological compound MCC950. In addition, while ROFA promoted the upregulation of pro-inflammatory gene expression and cytokines release, MCC950 reduced TNF-α, IL-6, and CCL2 production. Furthermore, inhibition of TNF-α with a neutralizing antibody decreased IL-1β release in ROFA-exposed BMDMs. Using electron tomography, ROFA particles were observed inside intracellular vesicles and mitochondria, which showed signs of ultrastructural damage. Mechanistically, we identified lysosomal rupture, K+ efflux, and impaired mitochondrial function as important prerequisites for ROFA-mediated IL-1β release. Interestingly, specific inhibition of superoxide anion production (O2•-) from mitochondrial respiratory Complex I, but not III, blunted IL-1β release in ROFA-exposed BMDMs. Our findings unravel the mechanism by which PM2.5 promotes IL-1β release in macrophages and provide a novel link between innate immune response and exposure to air pollution PM2.5.


Residual Oil Fly Ash (ROFA)
ROFA particles were collected as a by-product of fossil fuel combustion for power generation at Boston Edison Co., Mystic Power Plant (Mystic, CT, US) during burning of low-sulphur fuel oil N° 6 and were kindly provided by Dr. Godleski (Harvard School of Public Health, Boston, MA, US).ROFA samples used in the present work have an average aerodynamic diameter of 2.1 ± 1.6 μm and a high content of transition metals, namely Fe (III), Ni (II), and V (IV) as water soluble sulphates (Caceres et al., 2020).

Concentrated Ambient Particles (CAPs)
CAPs samples were collected using a Harvard Ambient Particle Concentrator (HAPC) located at the Harvard School of Public Health and were kindly provided by Dr. González-Flecha.CAPs size ranges from 0.1 to 2.5 μm and their elemental composition is predominantly defined by S and Si, with low transition metal content (Magnani et al., 2016).

Standard Reference Material (SRM) 1648a
SRM 1648a samples were collected from St. Louis City (MO, US) urban air and provided by the National Institute of Standards and Technology (NIST, Gaithersburg, MD, US).Morphological analysis indicates a mean particle diameter of 5.85 µm and elemental composition lists Fe as the most abundant transition metal.In addition, SRM 1648a has a high content of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) (SRM 1648a Certificate of Analysis).

SRM 2975
SRM 2975 samples are Diesel Exhaust Particles (DEP) collected from the exhaust of an industrial forklift from the M.E.Wright Donaldson Company Inc. (Minneapolis, MN, US) and provided by the NIST.Morphological and chemical composition analysis shows an average size of 1.62 ± 0.01 μm, and a high PAHs and nitro-PAHs content (SRM 2975 Certificate of Analysis).Elemental composition has been reported by (Farahani et al., 2021).

Human peripheral blood mononuclear cells (PBMCs) isolation, differentiation, and incubation
with ROFA Venous blood samples from 7 healthy donors (3 males and 4 females) were collected in BD Vacutainer CPT tubes and centrifuged at 1,800 g for 15 min at room temperature (22 ̊ C).Isolated PBMCs were transferred to a T25 flask, cultured at 37 °C and 5% CO2 in a humidified incubator, and differentiated in RPMI supplemented with recombinant human M-CSF (Immunotools GmbH, Friesoythe, Germany) at 100 ng/mL.After 6 days, monocyte-derived macrophages were plated in 96-well plates at a density of 1x10 5 cells/well and incubated with ROFA at 100 μg/mL or RPMI for 24 hours.Cell culture supernatants were stored at -80 °C until IL-1β detection by ELISA.

Cell viability
Cell viability was assessed by the CytoTox 96 assay (Promega, Madison, WI, US), which quantitatively measures lactate dehydrogenase (LDH) released in cell culture supernatants upon cytoplasmic membrane destabilization and cell lysis.

Flow cytometry
THP-1-ASC-GFP cells were acquired in a FACSCanto II equipment (BD Biosciences, Franklin Lakes, NJ, US) in the FL-1 channel.Gates for priming and specks formation were established according to THP-1-ASC-GFP relative distribution in the FITC-W and FITC-A channels.THP-1-ASC-GFP cells undergoing NLRP3 priming show enhanced green cytoplasmic fluorescence, thus a high FITC-A signal.Cells in the second stage of inflammasome activation display focalized fluorescence due to ASC-specks formation, thus showing a low FITC-W signal (Hoss et al., 2018).The same gating strategy was used when acquiring ASC-Citrine BMDMs.Data were analyzed by FlowJo Software version 10.8.1 (Tree Star, Ashland, OR, US).

Fluorescence microscopy
ASC-Citrine BMDMs were plated in 8-well chamber slides (Ibidi GmbH, Gräfelfing, Germany) at 5x10 4 cells/well and left overnight until achieving full cell adherence.Next, cells were incubated with ROFA at 100 μg/mL or RPMI for 6 or 24 hours.As a positive control for ASC-specks formation, ASC-Citrine BMDMs were incubated with 20 ng/mL LPS for 4 hours followed by stimulation with 5 μM nigericin (Invivogen) for 2 hours.Cell nuclei were counterstained with 1 μg/mL Hoechst dye.Images were acquired with a SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 63×/1.40oil objective.Bright field images were also acquired to evaluate BMDMs PM2.5 uptake.Data processing was performed with the LAS X Life Science software (Leica Microsystems).

qRT-PCR
To perform a time course analysis of NLRP3-related gene expression, wild type BMDMs were plated in 12-well plates at 5x10 5 cells/well and incubated with ROFA at 100 μg/mL or RPMI for 1, 3, 6, 12, and 24 hours.Additionally, samples from the 6-and 24-hour time point were used to build an inflammatory gene expression profile for PM2.5-exposed BMDMs.Total RNA was purified with the RNeasy Mini Kit (Qiagen, Düsseldorf, Germany) according to manufacturer's instructions.The High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, US) was used to obtain cDNA from 200 ng of total RNA.Targets of interest were amplified using Taq-Man probes (Supplementary Table 2) and a CFX96 Touch Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA).Target gene expression levels were quantified by the 2 -ΔΔCT method with β-actin as the housekeeping gene.

IL-1β and IL-18 detection
IL-1β was quantified by ELISA in cell culture supernatants from THP-1-ASC-GFP cells and human monocyte-differentiated macrophages using the Human IL-1β/IL-1F2 Quantikine ELISA Kit (R&D Systems) according to manufacturer's instructions.Cell culture supernatants from wild type, Nlrp3 -/-, and Casp1 -/-BMDMs were also analyzed for IL-1β content by ELISA using the Mouse IL-1β/IL-1F2 Quantikine ELISA Kit (R&D Systems) following manufacturer's recommendations.IL-18 was quantified by ELISA in cell culture supernatants from wild type BMDMs using the Mouse IL-18 DuoSet kit (R&D Systems) following manufacturer's recommendations.

Caspase-1 enzymatic activity
The Caspase-Glo 1 Inflammasome Assay Kit (Promega) was used for Caspase-1 activity assessment according to manufacturer's instructions.Wild type BMDMs were plated in 96-well plates at 1x10 5 cells/well and incubated with ROFA at 100 μg/mL or RPMI for 1, 3, 6, 12, and 24 hours.Cell culture supernatants were stored at -80°C until IL-1β detection by ELISA.Afterwards, cells were transferred to a white opaque plate and Caspase-1 activity-related chemiluminescent signal was followed for 60 min in an Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland).Data were analyzed as the ratio of the area under the curve (AUC) between RPMI and ROFA samples for each evaluated time point.

Cytokine detection
Cytokine levels were quantified by the CBA Mouse Inflammation Kit (BD Biosciences) in cell culture supernatants from wild type BMDMs according to manufacturer's instructions.

Electron tomography
BMDMs were plated in 6-well plates containing 6 mm sapphire discs at 5x10 6 cell/well and left overnight to achieve full cell adherence.Cells were incubated with ROFA at 100 μg/mL or RPMI for 6 or 24 hours.Samples were chemically fixed using isosmotic Karnovsky's fixative (2.4% sodium cacodylate, 0.75% paraformaldehyde, 0.75% glutaraldehyde), processed to Epon-Araldite resin, and imaged by dual-axis electron tomography as previously described (Rog-Zielinska et al., 2016).Imaging was performed at the Electron Microscopy Core Facility of the European Molecular Biology Laboratory (EMBL) (Heidelberg, Germany).Image reconstruction and segmentation were conducted using IMOD software (Rog-Zielinska et al., 2021).

Lysosomal disruption 10.1. Fluorescence microscopy
Wild type BMDMs were plated in 8-well chamber slides (Ibidi) at 5x10 4 cells/well and left overnight to achieve full cell adherence.Cells were incubated with ROFA at 100 μg/mL or RPMI for 6 or 24 hours and loaded with 1 μg/mL Acridine Orange for 20 min (Antunes et al., 2001).Maximum lysosomal rupture was achieved in wild type BMDMs incubated with 1.25 mM L-leucyl-L-leucine O-methyl ester (LLO-Me) for 10 min.Images were acquired with a Leica SP8 confocal microscope (Leica Microsystems) and data processing was performed with the LAS X Life Science software (Leica Microsystems).

Flow cytometry
After 6 or 24 hours of incubation with ROFA at 100 μg/mL or RPMI, wild type BMDMs were stained with 1 μM Acridine Orange for 20 min in the dark at 37 °C, and samples were acquired in a FACSCanto II equipment (BD Biosciences).Lysosomal rupture was followed by the loss of red fluorescence from the acidic lysosomal compartment in the PerCP-Cy5.5channel.Data were analyzed by FlowJo Software (Tree Star).

Inhibition of K + efflux
Wild type BMDMs were plated in 96-well plates at 1x10 5 cells/well and incubated with increasing extracellular K + concentration ([K + ]ex) in order to inhibit the electrochemical gradient that drives K + efflux (Gross et al., 2016).RPMI containing physiological [K + ]ex of 5 mM was used as a basal condition.In addition, RPMI was supplemented with 10, 20, 30, or 40 mM KCl to block K + efflux.After 6 or 24 hours of incubation with ROFA at 100 μg/mL or RPMI, cell culture supernatants were stored at -80 °C until IL-1β assessment by ELISA.
12. Mitochondrial function assessment 12.1.Mitochondrial respiration Oxygen consumption rate (OCR) was measured using a Seahorse XF96 Extracellular Flux Analyzer (Agilent, CA, US).BMDMs were seeded in 96-well plates at 8x10 4 cells/well in quadruplicates 6 hours before incubation with PM2.5 suspensions at 100 μg/mL or RPMI.After 6 or 24 hours, cell culture media was switched to bicarbonate-and phenol red-free RPMI (Gibco) containing 100 ng/mL recombinant human M-CSF, 10 mM glucose, and 2 mM glutamine, and incubated for 1 hour at 37 °C in the absence of CO2.Four baseline measurements and four response rates were measured, and the average of these rates was used for data analysis.Respiratory chain uncoupler and inhibitors were 1 µM FCCP, 1 µM Oligomycin A, 2 µM Antimycin A, and 2 µM Rotenone.OCR and indicators of mitochondrial bioenergetics were obtained using the Seahorse XF Cell Mito Stress Test Report Generator Software (Agilent) (Brand and Nicholls, 2011).Briefly, the Spare Respiratory Capacity indicates the capability of the cell to respond to an increased energetic demand, as well as how closely it is respiring at its theoretical maximum.The Coupling Efficiency is calculated from the change in basal OCR after Oligomycin addition and indicates the fraction of basal mitochondrial oxygen consumption used for ATP synthesis.The Proton Leak is the remaining basal OCR that is not coupled to ATP production, which may indicate damage to the inner mitochondrial membrane or be used as a mechanism to regulate ATP production.The Maximal Respiration represents the maximal OCR attained by adding FCCP, indicating the maximum rate of respiration that the cell can achieve.ATP Production indicates the ATP produced by the mitochondria that contributes to meeting the energetic needs of the cell.The decrease in OCR upon injection of Oligomycin represents the portion of basal respiration that was being used to drive ATP production.
12.2.Mitochondrial O2 •-production After 6 or 24 hours of incubation with PM2.5 suspensions at 100 μg/mL or RPMI, wild type BMDMs were incubated with 5 μM MitoSOX for 20 min in the dark at 37 °C.Samples were acquired in a FACSCanto II equipment (BD Biosciences).Mitochondrial O2 •-production was assessed as an increase in red fluorescence in the PE channel.Data were analyzed by FlowJo Software (Tree Star).

Statistics
Data are presented as mean ± SEM from at least three independent experiments.Unpaired Student's t-test was used to analyze the differences between two groups.One-way ANOVA followed by Dunnett's post hoc test or two-way ANOVA followed by uncorrected Fisher's test were performed to evaluate differences between more than two groups.Statistical significance was considered at p<0.05.